Inflammatory Cell Findings in the Female Rabbit Heart and Stress-associated Exacerbation with Handling and Procedures Used in Nonclinical Studies.

Despite the use of rabbits in biomedical research, including regulatory toxicology and cardiovascular studies, little data exist on heart findings in this species. This study was designed to document myocardial findings in female rabbits and the impact of study-related procedures typical for vaccine toxicology studies. One hundred and forty 6- to 8-month-old female New Zealand White rabbits were divided equally into 2 groups, high and low study procedure groups (group 1 and group 2, respectively). All animals received intramuscular (IM) injections of sterile saline every 2 weeks for 5 times and were necropsied 2 days after the final IM injection. Clinical chemistry, hematology, and urinalysis were evaluated. Blood for stress biomarkers (norepinephrine, epinephrine, cortisol, and corticosterone), C-reactive protein, cardiac troponin I, and creatine kinase were collected at time 0 (just before dose administration) and then at 4, 24, and 48 hr after dose administration in group 1 only. Hearts were assessed histologically. Focal to multifocal minimal inflammatory cell infiltrates were common (∼80%), particularly in the left ventricle and interventricular septum, and were similar to the types of infiltrates identified in other laboratory animal species. Additionally, study-related procedures elevated serum stress biomarkers and exacerbated the frequency and severity of myocardial inflammatory cell infiltrates.

Activation of TrkB with TAM-163 results in opposite effects on body weight in rodents and non-human primates.

Strong genetic data link the Tyrosine kinase receptor B (TrkB) and its major endogenous ligand brain-derived neurotrophic factor (BDNF) to the regulation of energy homeostasis, with loss-of-function mutations in either gene causing severe obesity in both mice and humans. It has previously been reported that peripheral administration of the endogenous TrkB agonist ligand neurotrophin-4 (NT-4) profoundly decreases food intake and body weight in rodents, while paradoxically increasing these same parameters in monkeys. We generated a humanized TrkB agonist antibody, TAM-163, and characterized its therapeutic potential in several models of type 2 diabetes and obesity. In vitro, TAM-163 bound to human and rodent TrkB with high affinity, activated all aspects of the TrkB signaling cascade and induced TrkB internalization and degradation in a manner similar to BDNF. In vivo, peripheral administration of TAM-163 decreased food intake and/or body weight in mice, rats, hamsters, and dogs, but increased food intake and body weight in monkeys. The magnitude of weight change was similar in rodents and non-human primates, occurred at doses where there was no appreciable penetration into deep structures of the brain, and could not be explained by differences in exposures between species. Rather, peripherally administered TAM-163 localized to areas in the hypothalamus and the brain stem located outside the blood-brain barrier in a similar manner between rodents and non-human primates, suggesting differences in neuroanatomy across species. Our data demonstrate that a TrkB agonist antibody, administered peripherally, causes species-dependent effects on body weight similar to the endogenous TrkB ligand NT-4. The possible clinical utility of TrkB agonism in treating weight regulatory disorder, such as obesity or cachexia, will require evaluation in man.

Agonistic TAM-163 antibody targeting tyrosine kinase receptor-B: applying mechanistic modeling to enable preclinical to clinical translation and guide clinical trial design.

TAM-163, an agonist monoclonal antibody targeting tyrosine receptor kinase-B (TrkB), is currently being investigated as a potential body weight modulatory agent in humans. To support the selection of the dose range for the first-in-human (FIH) trial of TAM-163, we conducted a mechanistic analysis of the pharmacokinetic (PK) and pharmacodynamic (PD) data (e.g., body weight gain) obtained in lean cynomolgus and obese rhesus monkeys following single doses ranging from 0.3 to 60 mg/kg. A target-mediated drug disposition (TMDD) model was used to describe the observed nonlinear PK and Emax approach was used to describe the observed dose-dependent PD effect. The TMDD model development was supported by the experimental determination of the binding affinity constant (9.4 nM) and internalization rate of the drug-target complex (2.08 h(-1)). These mechanistic analyses enabled linking of exposure, target (TrkB) coverage, and pharmacological activity (e.g., PD) in monkeys, and indicated that ≥ 38% target coverage (time-average) was required to achieve significant body weight gain in monkeys. Based on the scaling of the TMDD model from monkeys to humans and assuming similar relationship between the target coverage and pharmacological activity between monkey and humans, subcutaneous (SC) doses of 1 and 15 mg/kg in humans were projected to be the minimally and the fully pharmacologically active doses, respectively. Based on the minimal anticipated biological effect level (MABEL) approach for starting dose selection, the dose of 0.05 mg/kg (3 mg for a 60 kg human) SC was recommended as the starting dose for FIH trials, because at this dose level<10% target coverage was projected at Cmax (and all other time points). This study illustrates a rational mechanistic approach for the selection of FIH dose range for a therapeutic protein with a complex model of action.

All-trans retinoic acid antagonizes the action of calciferol and its active metabolite, 1,25-dihydroxycholecalciferol, in rats.

An antagonistic interaction between retinol and calciferol has been established. However, the mechanism by which this antagonism occurs is unclear. One possibility is that retinol affects the metabolism of calciferol. To investigate this hypothesis, retinol- and calciferol-depleted rats were given various amounts of ergocalciferol, cholecalciferol, 1alpha,25-dihydroxycholecalciferol [1,25(OH)2D3], or 24,24-difluoro-1alpha,25-dihydroxycholecalciferol [24-F2-1,25(OH)2D3] in combination with various amounts of retinyl acetate or all-trans retinoic acid (ATRA) in a series of studies. Rats administered 1720 or 3440 microg retinyl acetate once every 3 d for 33 d in combination with 25.8 ng ergocalciferol or 25 ng cholecalciferol every 3 d had lower serum calcium and greater serum phosphorus concentrations than rats fed 0 or 11.4 mug retinyl acetate every 3 d. In addition, rats fed 400 microg ATRA/d in combination with 25.8 ng ergocalciferol every 3 d, 25 ng cholecalciferol every 3 d, 2-5 ng 1,25(OH)2D3/d, or 0.5-1 ng 24-F2-1,25(OH)2D3/d had significantly lower serum calcium and higher serum phosphorus concentrations than rats not given ATRA in the diet. Therefore, both retinyl acetate and ATRA are able to antagonize the action of ergocalciferol and cholecalciferol in vivo. Additionally, ATRA antagonizes the in vivo action of 1,25(OH)2D3 and an analog, 24-F2-1,25(OH)2D3, that cannot be 24-hydroxylated. Together, these results suggest that retinol does not antagonize the action of calciferol by altering the metabolism of calciferol or 1,25(OH)2D3, but does so by another mechanism.

Bone resorption activity of all-trans retinoic acid is independent of vitamin D in rats.

The mechanism by which all-trans retinoic acid (ATRA) induces bone resorption is unknown. However, an interaction between vitamin A and vitamin D has been established. In fact, although the mechanism is still unclear, vitamin A has been shown to be a weak antagonist of the actions of vitamin D. Taking into account this interaction and the influence of vitamin D on other calcitropic hormones, such as parathyroid hormone, the effect of vitamin D on ATRA-induced bone resorption was investigated. Vitamin D-deficient rats were fed diets containing 0 or 150 micro g of ATRA/g of diet. The rats then were orally administered 0 or 625 ng of cholecalciferol (vitamin D(3)) daily. Various bone parameters were measured after 3-8 wk. Regardless of the presence or absence of vitamin D(3), ATRA was able to cause bone resorption. In addition to examining the effect of vitamin D on ATRA-induced bone resorption under normal conditions, this effect also was studied under conditions that inhibit bone mineralization or growth by altering dietary calcium (Ca) and phosphorus (P) levels. Changes in dietary levels of Ca and P did not affect the ability of ATRA to cause bone resorption. Interestingly, despite its ability to stimulate bone resorption, ATRA did not affect serum calcium or phosphorus levels. Overall, the ability of ATRA to cause bone resorption is not dependent on vitamin D(3), dietary Ca or dietary P.